1. Field of the Invention
The present invention relates generally to the field of diagnostic tests and, more particularly, to those tests useful in qualitative and quantitative determination of biological components, such as lactic acid and ketone bodies, in which tests such components are converted to an oxidizing substance, such as a peroxide.
2. Description of the Prior Art
The technique of using a tetrazolium compound and an electron-carrying intermediary (such as phenazine methosulfate or diaphorase) to turn reduced nicotine adenine dinucleotide (phosphate) [NAD(P)H] into color has been known for some time. Markert, C. L. and Moller, F., Proc. Nat. Acad. Sci. U.S. 45, 753 (1959); Tsao, M. U., Arch. Biochem. Biophys., 90, 234 (1960); Dewey, M. M. and Conklin, J. L.,Proc. Soc. Exp. Biol. and Med. 105, 492 (1960); Nachlas, M. M., Margulies, S. I., Goldberg, J. D., and Seligman, A. M., Anal. Biochem. 1, 317 (1960); Babson, A. L. and Phillips, G. E., Clin. Chim. Acta 12, 210 (1965); Gay, R. J., McComb, R. B., and Bowers, G. N., Clin. Chem. 14, 740 (1968). However, this technique suffers from the problems of phenazine methosulfate light sensitivity, insolubility of the formazan dye product, and diaphorase insolubility and instability.
The reaction of NAD(P)H with copper sulfate yields a cuprous ion that can then form a chelation product into neocuproin. This chelation product is colored. Morgenstern, S., Flor, R., Kessler, G., and Klein, B., Anal. Biochem 13, 149 (1965).
Reduced nicotine adenine dinucleotide (NADH) reacts with 2-oxobutyrate and 2,4-dinitrophenylhydrazine in the presence of lactate dehydrogenase to form 2,4-dinitrophenylhydrazone. King, J., Practical Clinical Enzymology, D. Van Nostrand Co., Ltd., p. 55, (1965); Cabaud, P. G. and Wroblewski, F., Am. J. Clin. Path. 30, 234 (1958). This product is colored. However, this reaction requires two separate discrete steps for color generation. Moreover, the accuracy of the method has been questioned. Massod, M. F., Franey, R. J., Therrien, M. E., Rideout, P. T., and Babcock, M. T., Am. J. Clin. Path. 42, 623 (1964).
Evidence for the existence of an uncoupling hydroxylase was first found in 1961 by Kaufman. Kaufman, S., Biochim. Biophys. Acta 51, 619 (1961). The nonstoichiometric nature of the hydroxylase reaction indicated that some product, other than hydroxylated substrate, was being produced. Since that time many other examples of this phenomenon have been seen. Most uncoupling hydroxylases are not completely uncoupled. In other words, there is some hydroxylated product formed and some hydrogen peroxide formed. A few can be completely uncoupled by certain pseudosubstrates. In other words, there is no hydroxylated product formed. An example of a 100% uncoupled system is salicylate hydroxylase and benzoate. White-Stevens, R. H. and Kamin, H., J. Biol. Chem. 247, 2358 (1972); White-Stevens, R. H., Kamin, H. and Gibson, Q. H., J. Biol. Chem. 247, 2371 (1972); White-Stevens, R. H., Kamin, H., and Gibson, Q. H. in "Oxidation Reduction Enzymes", Akeson, A. and Ehrenberg, A., eds., p. 453, Pergamon Press, Oxford and New York (1972); White-Stevens, R. H. and Kamin, H. Biochem. and Biophys. Res. Comm. 38, 882 (1970). Other examples of completely or partially uncoupled hydroxylases include phenylalanine hydroxylase, Storm, C. B., and Kaufman, S., Biochem and Biophys. Res. Comm. 32, 788 (1968); Fisher, D. B. and Kaufman, S., J. Biol. Chem. 248, 4300 (1973); p-hydroxybenzoate hydroxylase, Spector, T. and Massey, V., J. Biol. Chem. 247, 4679 (1972); Howell, L. G., Spector, T., and Massey, V., J. Biol. Chem., 247, 4340 (1972); Howell, L. G. and Massey, V., Biochem. and Biophys. Res. Comm. 40, 887 (1970); and orcinol hydroxylase, Ohta, Y., Higgins, I. J., and Ribbons, D. W., J. Biol. Chem. 250, 3814 (1975).
There has been only one publication in which the hydroxylase system has been linked to peroxidase. This was done for the purpose of detecting hydrogen peroxide formation rather than for the assay of any component taking part in the reactions. Storm, C. B., and Kaufman, S., supra. Peroxidase was used to oxidize a reduced cofactor that was part of the hydroxylase reaction. The altered ratio of hydroxylated product formed indicated that hydrogen peroxide had been present. This reaction did not involve the formation of a colored product. The object of the experiment was to demonstrate that the hydroxylase reaction had generated some H.sub.2 O.sub.2. No reference has been found that discloses the use of uncoupling hydroxylases, either alone or in combination with a peroxidase system, as an assay or a detection method.